Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers Stephanie M. Cope, Sandip Shinde, Robert B. Best, Giovanna Ghirlanda, Sara M. Vaiana  Biophysical Journal  Volume 105, Issue 7, Pages 1661-1669 (October 2013) DOI: 10.1016/j.bpj.2013.08.026 Copyright © 2013 The Authors Terms and Conditions

Figure 1 Sequences of human IAPP (hIAPP), rat IAPP (rIAPP), and N_loop. The disulfide bond between residues 2 and 7, forming a loop at the N-terminus of the sequences, is indicated by the blue brackets. All sequences have a free N-terminus and amidated C terminus. To see this figure in color, go online. Biophysical Journal 2013 105, 1661-1669DOI: (10.1016/j.bpj.2013.08.026) Copyright © 2013 The Authors Terms and Conditions

Figure 2 (a) Oxidized N_loop fibers, visible immediately upon dissolving peptide in buffer (50 mM peptide in 50 mM sodium acetate, pH 4.9). Immediate formation of fibers was also observed at concentrations of 57 μM by TEM (Fig. S1). (b) Optical microscopy of N_loop fibers in the absence (left) and presence (right) of crossed polarizers, revealing fiber birefringence (scale bar, 1 mm). (c) TEM image of fibers prepared at 25 mM (scale bar, 200 nm). Fibers of same morphology were observed at concentrations as low as 57 μM (Fig. S1). To see this figure in color, go online. Biophysical Journal 2013 105, 1661-1669DOI: (10.1016/j.bpj.2013.08.026) Copyright © 2013 The Authors Terms and Conditions

Figure 3 (a) CD spectra of linear N1–8 (gray line) and cyclic N_loop (black line) peptides at 5°C compared to pure component spectra for disordered coil (dotted gray line), type I β-turn (dotted black line), and type II β-turn (dotted blue line) (redrawn from Perczel and Fassman (30)); MRE, mean residue ellipticity. (b) Temperature dependence of CD spectra from 5°C to 70°C of N_loop and N1–8 (inset) (660 μM peptide in 50 mM sodium acetate, pH 4.9). (c) Concentration dependence of N_loop CD signal amplitude at minimum, ε, rescaled by pathlength l (solid circles) and linear fit (line); renormalized CD spectra do not vary with concentration (inset) (20°C, pH 4.9 in 10–25 mM sodium acetate buffer). (d) FTIR spectra of KBr pellets of HPLC-purified N_loop peptide (gray) versus fibers (red) in the amide region. N_loop samples were lyophilized immediately after HPLC purification. N_loop fiber samples were lyophilized after 1 week aging at 4°C (7 mM peptide in 50 mM NaAc, pH 4.9). Peak positions for the amide I (1675 cm−1) and II (1533 cm−1) bands are given for the N_loop spectrum. The vertical gray line indicates the characteristic amide I peak of IAPP amyloid fibrils. All spectra are atmospheric compensated, baseline corrected, and KBr subtracted. To see this figure in color, go online. Biophysical Journal 2013 105, 1661-1669DOI: (10.1016/j.bpj.2013.08.026) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Comparison of secondary chemical shifts measured for N_loop (green), using random coil values of reference De Simone et al. (23), and values reported in Yonemoto et al. (16) and Williamson and Miranker (43), for residues 1–8 of full-length hIAPP (red) and rIAPP (blue). Values computed from REMD simulations using SPARTA+ (black) are also compared with experimental data sets. Error bars on the calculated shifts represent the root mean-squared error in the shift prediction algorithm. To see this figure in color, go online. Biophysical Journal 2013 105, 1661-1669DOI: (10.1016/j.bpj.2013.08.026) Copyright © 2013 The Authors Terms and Conditions

Figure 5 Structure of N_loop from molecular simulations in CPK representation. (a) Most populated conformation (64.0%), with type I turn between residues 3 and 6. (b) Next-most populated conformation (33.3%), with type I turn between residues 4 and 7. To see this figure in color, go online. Biophysical Journal 2013 105, 1661-1669DOI: (10.1016/j.bpj.2013.08.026) Copyright © 2013 The Authors Terms and Conditions